Trehalose—A nonpermeable cryoprotectant for direct freezing of early stage murine embryos

Krag, K.T.; Koehler, I-M.; Wright, Raymond W.

doi:10.1016/0011-2240(85)90134-8

Cited by 4 publications

(2 citation statements)

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“…The rapid freezing of embryos has usually been done by direct plunging into liquid nitrogen (Takeda et al, 1984;Krag et al, 1985;Biery et al, 1986;Trounson et al, 1987Reichenbach and Rodrigues, 1988) or into liquid nitrogen vapor Kanagawa, 1985, 1988;Shelton, 1986a,b, 1987;Williams and Johnson, 1986), but, during direct plunging into liquid nitrogen, straws are sometimes broken. We have used the cooling system described in the present text to prevent the breakage of straws and to keep the cooling rate constant Kanagawa, 1985, 1988).…”

Section: Discussionmentioning

confidence: 99%

“…The former freezing procedure requires only a brief, one-step exposure of embryos to the cryoprotectant solution at room temperature, followed by direct plunging into liquid nitrogen. Mouse embryos can be frozen in glycerol plus sucrose (Takeda et al, 1984;Biery et al, 1986;Williams and Johnson, 1986;Shelton, 1986a,b, 1987;Reichenbach and Rodrigues, 1988); trehalose (Krag et al, 1985); raffinose, lactose, or glucose (Takahashi and Kanagawa, 1985); dimethyl sulfoxide plus sucrose (Trounson et al, 1987, 19881; and 1, 2-propanediol plus sucrose (Sathananthan et al, 1988). Regarding the exposure to cryoprotectant solution, however, only Szell and Shelton (1986b) have demonstrated that the effect of equilibration at 20°C in glycerol-sucrose mixture was not significant, and these authors concluded that complete equilibration was not necessary.…”

Section: Introductionmentioning

confidence: 99%

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Effect of equilibration period on the viability of frozen‐thawed mouse morulae after rapid freezing

Takahashi

Kanagawa

1990

Molecular Reproduction Devel

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Mouse morulae were frozen with 1.5-4.0 M glycerol + 0.25 M lactose solution by direct plunging into liquid nitrogen vapor 0.5-30 min after equilibration at room temperature. After thawing, embryos were cultured in vitro, and the highest survival rates were obtained after exposure for 3 min at 3.0 and 4.0 M and for 5 min at 1.5 and 2.0 M glycerol levels. Significant reductions in the survival rates (P less than 0.05) were observed when equilibration periods were extended for 3-5 min at 3.0 and 4.0 M and for 5-10 min at 1.5 and 2.0 M glycerol levels. These results clearly demonstrate that the equilibration time of embryos in glycerol-lactose mixture is one of the most important factors in the present rapid freezing conditions. To clarify the factors that lower embryo viability after prolonged equilibration, we performed further experiments on the effects of exposure to glycerol-lactose mixture on the developmental potential of embryos without freezing and on the volume changes of embryos during the exposure to glycerol solution with or without lactose. It was suggested that the detrimental effects of prolonged equilibration are due not only to the toxicity and osmotic injury of higher concentrations of cryoprotectant solution but also to the influx of water into embryonic cells caused by the hypotonic salt concentration of the extracellular (freezing) solution.

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